Persistence of procoagulant surface expression on activated human platelets: involvement of apoptosis and aminophospholipid translocase activity.

BACKGROUND: Activated platelets express a procoagulant surface when the asymmetric distribution of membrane phospholipids is scrambled, leading to phosphatidylserine (PS) exposure. PS expression, associated with apoptosis in nucleated cells, would be expected to be reversed by aminophospholipid translocase (APLT) activity. OBJECTIVE: To determine whether the procoagulant surface of activated platelets persists after it forms; to examine whether PS expression on platelets is associated with loss of mitochondrial inner membrane potential (DeltaPsi(m)), a hallmark of apoptosis; and to investigate the role of APLT in persistence of PS expression. METHODS: Platelets were stimulated with thrombin, collagen, a combination of both, or the Ca(2+)-ionophore A23187. Up to 4 h after activation, procoagulant surface expression was measured by annexin A5 binding by flow cytometry and by a prothrombinase assay. Flow cytometry was also used to measure PS expression concurrently with DeltaPsi(m) collapse, using CMXRos. APLT activity in annexin A5-negative and -positive platelets was measured flow cytometrically as the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. RESULTS AND CONCLUSIONS: Procoagulant surface expression on activated platelets persisted in vitro for at least 4 h; if such persistence occurs in vivo, there are important implications for the propagation of thrombosis. With the physiological stimuli, only 10-20% of the activated platelets expressed PS on their surface, and of these, only a portion exhibited DeltaPsi(m) collapse, indicating that PS expression can be associated with platelet apoptosis, but can also occur independently. APLT activity was very low in the PS-expressing platelet subpopulation for up to 4 h after activation, indicating that the persistence of a procoagulant surface may be attributed, at least in part, to this reduced APLT activity.

Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence.

BACKGROUND: The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (DeltaPsim), have been implicated in platelet clearance. OBJECTIVE: To investigate whether shortened platelet survival and steady-state platelet senescence are associated with increased surface exposure of PS and DeltaPsim collapse. METHODS: Survival of in-vitro biotinylated rabbit platelets treated with thrombin or Ca(2+)-ionophore A23187 was tracked by flow cytometry after injection. Steady-state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and DeltaPsim of in-vitro biotinylated platelets and of the aging platelet cohort biotinylated in-vivo were measured by flow cytometry using annexin V-FLUOS and the DeltaPsim-sensitive dye CMXRos, respectively. RESULTS: Although PS expression, DeltaPsim and survival of thrombin-degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only one-sixth of PS-expressing platelets also exhibited DeltaPsim loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed DeltaPsim; of the 17% of PS-expressing platelets, one-third did not exhibit DeltaPsim loss. There was also an increase in platelets with collapsed DeltaPsim but not expressing PS. CONCLUSIONS: Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of DeltaPsim collapse and conversely, in aged platelets, DeltaPsim loss can occur independently of PS expression.